In-Situ Probe

ABSTRACT

A device for monitoring a bioreactor is configured for in-situ analysis, e.g., by NIR, without the need for withdrawing a sample into a sample cell or into an ex-situ arrangement. The device can be inserted into a port of the bioreactor and provides a sample detection region defined by an optical element such as a lens and a photodetector. The electrical signal obtained from a photodetector that is part of the device can be directed to an analyzer via a detachable electrical connection.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.17/030,032, filed on Sep. 23, 2020, which claims the benefit under 35USC 119(e) of U.S. Provisional Patent Application No. 62/904,560, filedon Sep. 23, 2019 and of U.S. Provisional Patent Application No.63/012,532, filed on Apr. 20, 2020, all of which are incorporated hereinby this reference in their entirety.

BACKGROUND OF THE INVENTION

Many processes in the chemical, biochemical, pharmaceutical, food,beverage and in other industries require some type of monitoring.

Sensors have been developed and are available to measure pH, dissolvedoxygen (DO), temperature or pressure in-situ and in real-time. Commontechniques for detecting chemical constituents include high performanceliquid chromatography (HPLC), gas chromatography-mass spectroscopy(GCMS), or enzyme- and reagent-based electrochemical methods.

While considered accurate, many existing approaches for monitoringsubstances in a reactor are conducted off-line, tend to be destructivewith respect to the sample, often require expensive consumables and/ortake a long time to complete. In many cases, the equipment needed toperform these analyses is expensive, requires involved calibrations, andtrained operators. Procedures may be time- and labor-intensive, oftenmitigated by decreasing the sampling frequency of a given process, thusreducing the data points. Often, samples are run in batches, after theprocess has been completed, yielding little or no feedback for adjustingconditions on an ongoing basis. Drawbacks such as these can persist evenwith automated sampling operations.

Various optical spectroscopy approaches are available to assesscomponents, also referred to as analytes, in a sample. Among these,probably the most common is absorption spectroscopy. Incident lightexcites electrons of the analyte from a low energy ground state into ahigh energy, excited state, and the energy can be absorbed by bothnon-bonding n-electrons and it-electrons within a molecular orbital.Absorption spectroscopy can be performed in the ultraviolet, visible,and/or infrared region, with analytes of varying material phases andcomposition being interrogated by specific wavelengths or wavelengthbands of light. The resulting transmitted light is then used to resolvethe absorbed spectra, to determine the analyte's or sample'scomposition, temperature, pH and/or other intrinsic properties forapplications ranging from medical diagnostics, pharmaceuticaldevelopment, food and beverage quality control, to list a few.

Another option is Raman spectroscopy, which works by the detection ofinelastic scattering of typically monochromatic light from a laser.

SUMMARY OF THE INVENTION

A need exists for robust, hands-free, non-destructive, real timetechniques for identifying and/or quantifying constituents in a givenprocess. Typically, the process is conducted in a vessel, e.g., abioreactor. The contents of the bioreactor can change as the processunfolds and data obtained by the procedures and equipment describedherein can be used to monitor, adjust and/or control process parameters.

In many of its aspects, the invention relates to a device and/or methodfor monitoring, in-situ, an ongoing process, such as, for example, aprocess conducted in a bioreactor. Cells and/or substances present inthe bioreactor (or another vessel) are identified and often quantifiedusing a suitable technique. In many implementations, the technique isnear infrared (NIR) absorption spectrometry.

Some embodiments relate to an in-situ probe that can be inserted and/ormaintained in a bioreactor and incorporates elements for interrogatingas well as elements needed to analyze the contents of a bioreactor,e.g., in the NIR region of the electromagnetic spectrum. The analysiscan be conducted in real time, in a nondestructive manner. Otherembodiments relate to a system that includes a bioreactor monitored byan in-situ probe, in an automated manner, for example. Furtherembodiments relate to a method for performing an in-situ analysis usingan in-situ probe.

In typical examples, the analysis is conducted without withdrawing asample from the reactor. An interrogating beam of electromagneticradiation is introduced into the reactor and traverses a pathlengthformed within the reactor medium; transmitted radiation reaches aphotodetector and generates an electrical signal that is conveyed andanalyzed externally.

In general, according to one aspect, the invention features a device formonitoring a vessel. This device comprises an outer tube and a tipsection at the end of the tube. The tip section has a sample detectionregion defined by an optical transmission port and an optical detectionport, wherein light received through the optical detection port isdetected by a photodetector housed in the tip section.

In examples, the device further comprises an inner tube housed in theouter tube for providing the light for the optical transmission port.This inner tube contains electrical wiring for the photodetector. Insome examples, the inner tube contains optical fiber for carrying thelight for the optical transmission port. In other examples, the innertube defines a free space path for the light for the opticaltransmission port.

In many embodiments, a housing carried by the inner tube makeselectrical connections to the optical detector.

A fitting on the outer tube is common for sealing with a port of abioreactor.

Then, in operation, the light is generated by a tunable laser thatsweeps a narrow band emission across an infrared spectral band.

In many cases, a focusing lens in the tip section is helpful forconditioning, i.e., focusing or collimating, the light transmittedacross the sample detection region.

In general, according to another aspect, the invention features a methodfor monitoring a vessel. The method comprises inserting an outer tubewith a tip section into the vessel, transmitting light across a sampledetection region of the tip section from an optical transmission port toan optical detection port, and detecting the light received through theoptical detection port with a photodetector housed in the tip section.

Often, the method further includes autoclaving the outer tube includingthe tip section prior to inserting the outer tube into the vessel.

Then, after inserting the outer tube into the vessel, an inner tube isinserted in some examples. This inner tube includes a housing into theouter tube.

In general, according to another aspect, the invention features a systemcomprising an in-situ probe inserted in a port of a reactor, wherein theprobe includes a photodetector for receiving electromagnetic radiationthat has propagated through a sample detection region defined by thephotodetector and an optical element, and a controller for analyzing anelectrical signal from the photodetector and including a lasergenerating the electromagnetic radiation.

In general, according to another aspect, the invention features a systemcomprising a bioreactor and a device for monitoring in-situ contents inthe bioreactor. This device includes an outer tube inserted through aport of the bioreactor, a tip section at the end of the tube, the tipsection having a sample detection region defined by an opticaltransmission port and an optical detection port, wherein light receivedthrough the optical detection port is detected by a photodetector housedin the tip section.

In general, according to another aspect, the invention features a methodfor monitoring a reactor. The method comprises directing a beam ofelectromagnetic radiation through a sample detection region within areactor medium, wherein the sample detection region is defined by a lensand a photodetector, collecting an electrical signal from thephotodetector, and analyzing the electrical signal to obtain a spectrumof a material in the sample detection region.

The in-situ probe, system and method present many advantages. Detachableparts that can be assembled and disassembled as needed offer flexibilityand convenience. In many cases, the analysis process is simplified andaccelerated. Moreover, the probe can include disposable parts and/or,importantly, components that can be autoclaved and reused.

Whereas many existing approaches rely on removing and/or circulatingcells in loops external to the process vessel, typically through apumping system, the equipment and procedures described herein reduce,minimize and often eliminate the exposure of the bioreactor contents toconditions external to the bioreactor. In addition, cells are preventedfrom being drawn into the pumping system.

Techniques such as the ones described herein also improve the quality ofthe analysis. For example, embodiments described herein can provideimproved or even maximum signal to noise ratios. This is accomplished bylaunching a light beam straight out of a fiber and/or a free space link,through a sample gap, with the transmitted light impinging onto aphotodetector. By having the detector cables running up the length ofthe probe, rather than using a return fiber optic cable leading to aphotodiode, often external to the reactor, approaches described hereincan rely on the signal to noise ratio (SNR) of the electrical cables,which generally are superior to fiber optics.

The above and other features of the invention including various noveldetails of construction and combinations of parts, and other advantages,will now be more particularly described with reference to theaccompanying drawings and pointed out in the claims. It will beunderstood that the particular method and device embodying the inventionare shown by way of illustration and not as a limitation of theinvention. The principles and features of this invention may be employedin various and numerous embodiments without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, reference characters refer to the sameparts throughout the different views. The drawings are not necessarilyto scale; emphasis has instead been placed upon illustrating theprinciples of the invention. Of the drawings:

FIGS. 1 and 2 are, respectively, perspective and side views of oneembodiment of an in-situ probe according to the invention.

FIG. 3 is a cross-sectional view of the tip section of the in-situ probeof FIGS. 1 and 2 according to one embodiment.

FIGS. 4A and 4B are views of the alignment elements used in assemblingthe in-situ probe of FIG. 1 .

FIGS. 5A and 5B are diagrams illustrating the assembly of anautoclavable component of the in-situ probe of FIG. 1 .

FIG. 6 is a diagram illustrating the assembly of the outer and innertubes to produce the in-situ probe of FIG. 1 .

FIGS. 7 and 8 are, respectively, isometric and side views of anotherembodiment of an in-situ probe according to the invention.

FIG. 9 is a cross-sectional view of the in-situ probe of FIGS. 7 and 8 .

FIG. 10 is a cross-sectional of the tip section of the in-situ probe ofFIGS. 1 and 2 .

FIGS. 11A and 11B are diagrams illustrating the preparation of anautoclavable component of the in-situ probe of FIGS. 7 and 8 .

FIG. 12 is a diagram illustrating the assembly of the in-situ probe ofFIGS. 7 and 8 .

FIG. 13 is a diagram of a system in which a stirred tank reactor isprovided with an in-situ probe according to embodiments of theinvention.

FIG. 14 is a diagram of a rocking bioreactor monitored by an in-situprobe controlled by a controller.

FIG. 15 is a series of plots showing viable cell densities under varioussampling and analysis conditions.

FIG. 16 shows plots in which samples of increasing cell densities arescanned in the NIR wavelengths, leading to increased scatter of the beamand thus an apparent increased absorbance.

FIG. 17 shows a comparison of cell density versus time measured bycytometry and NIR spectrometry.

FIG. 18 presents a comparison of samples of Pichia growing in a shakeflask. NIR measurements were performed with an in-line probe takingsamples automatically and reading absorbance at approximately 1450 nmand comparing to off-line measurements from a standardspectrophotometer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention now will be described more fully hereinafter withreference to the accompanying drawings, in which illustrativeembodiments of the invention are shown. This invention may, however, beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items. Further, the singular formsand the articles “a”, “an” and “the” are intended to include the pluralforms as well, unless expressly stated otherwise. It will be furtherunderstood that the terms: includes, comprises, including and/orcomprising, when used in this specification, specify the presence ofstated features, integers, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, integers, steps, operations, elements, components,and/or groups thereof. Further, it will be understood that when anelement, including component or subsystem, is referred to and/or shownas being connected or coupled to another element, it can be directlyconnected or coupled to the other element or intervening elements may bepresent.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

Many processes conducted in bioreactors require or benefit from astringent control of parameters such as pH, levels of oxygen, nutrients,metabolites and/or other species. In many of its aspects, the inventionrelates to a device and method for analyzing the contents of abioreactor on an ongoing basis. Cells, for instance, and/or otherreactor constituents can be detected, at various time intervals and thedata can be used to assess conditions and, if necessary, adjust oroptimize process parameters. Examples of processes that can be monitoredinclude cell growth protocols, fermentations, and so forth. Bioreactorscan feature a suitable design and can be characterized by a specificvolume or dimensions, as known in the art or as developed in the future.

In one implementation, techniques described herein are practiced with abioreactor that houses or is a cell culture system for thethree-dimensional assembly, growth and differentiation of cells andtissues. The bioreactor can contain cells, culture media, nutrients,metabolites, enzymes, hormones, cytokines and so forth.

Analysis can utilize a spectroscopy system for determining the spectralresponse of the components in the sample cell in one or more of thefollowing electromagnetic spectral regions: millimeter, microwave,terahertz, infrared (including near-, mid- and/or far-infrared),visible, ultraviolet (UV) , x-rays and/or gamma rays. Further, thespectroscopy system can measure different characteristics, such asabsorption spectra, emission (including blackbody or fluorescence)spectra, elastic scattering and reflection spectra , impedance (e.g.,index of refraction) spectra, and/or inelastic scattering (e.g., Ramanand Compton scattering) spectra, of analytes in the bioreactor.

Specific embodiments described herein rely on spectroscopy in theultraviolet and visible regions, but mostly in the infrared regionextending from 700 nanometers (nm) to 1 millimeter (mm) in wavelengthand specifically including the near infrared (0.75-1.4 μm, NIR),short-wavelength infrared (1.4-3 μm, SWIR), mid-wavelength infrared (3-8μm, MWIR), long-wavelength infrared (8-15 μm, LWIR), and the farinfrared (15-1000 μM, FIR) of the spectrum,. Probing molecular overtoneand combination vibrations, NIR-SWIR spectroscopy covers the region offrom 780 nanometer (nm) to 2500 nm of the electromagnetic spectrum. Anoverview of NIR spectroscopy can be found, for example, in an article byA.M.C. Davies in “An Introduction to Near Infrared (NIR) Spectroscopy”,http://www.impublications.com/content/introduction-near-infrared-nir-spectroscopy.See also, Cervera, A. E., Petersen, N., Lantz, A. E., Larsen, A. &Gernaey, K. V. Application of near-infrared spectroscopy for monitoringand control of cell culture and fermentation, Biotechnol. Prog. 25,1561-1581 (2009); and Roggo Y, et al., “A review of near infraredspectroscopy and chemometrics in pharmaceutical technologies”, Journalof Pharmaceutical and Biomedical Analysis, Volume 44, Issue 3, 2007.

Among its strength, infrared spectroscopy presents a non-invasive,non-destructive investigative approach, typically involving fast scantimes. A discussion of NIR as applied to microfluidic and other systemsis provided in U.S. patent application Ser. No. 16/419,690, to Hassellet al., filed on May 22, 2019 and incorporated herein in its entirety bythis reference.

U.S. Provisional Patent Application No. 62/892,702, to Hassell et al,filed on Aug. 28, 2019 under the title Device and Bioreactor MonitoringSystem and Method and incorporated herein by this reference in itsentirety, describes arrangements and techniques for obtaining samples tobe analyzed, e.g., by NIR, using a sample tube that can be inserted intoa vessel, e.g., a bioreactor or another type of vessel or arrangementused to conduct biochemical or chemical processes. The sample tube usedfor extracting a sample from the bioreactor can be combined orintegrated with a sample cell configured for NIR interrogation andanalysis.

According to embodiments described herein, measurements are taken within(inside) the reactor, typically without a need to withdraw a sample intoa sample or flow cell or into an external (ex-situ) arrangement fortaking a reading.

Many implementations of the present invention employ an outer tube ofstainless steel which is sealed by a photodetector, such as a photodiodein a windowed package, and an optical component, in many cases afocusing lens. These two elements define an optical transmission portand an optical detection port on either side of a sample detectionregion. Being in contact with the fluid inside the bioreactor, theirspacing can be the pathlength of the laser light.

In other embodiments, the photodetector and the optical component arebehind respective windows for ease of cleaning, however.

In practice, the outer tube is placed into a bioreactor and then anotherstainless steel cap with O-ring can be attached on top to create a seal.

This entire assembly can be autoclaved.

After autoclaving, the cap is removed. An inner tube, containing, forexample, the fiber optic collimator, which guides the beam downward intothe focusing lens, as well as a connector, e.g., a 3-pin POGO connector,which connects to the other connectors from the photodetector can beinserted into the outer tube. Some designs do not employ a collimatorand use the inner tube to form a free space path to propagate the beamof electromagnetic radiation towards the focusing lens.

One embodiment of an in-situ probe according to aspects of the inventionis described with reference to FIGS. 1-6 .

Shown in FIGS. 1 and 2 is in-situ probe 10, configured for placingand/or maintaining a sample detection region 12 within the bioreactor.FIG. 3 is a longitudinal cross-sectional view of a tip section 14 of thein-situ probe.

As seen in these figures, the sample detection region 12 is located at atip section 14 of probe 10 and can be shaped with an indentation formed,as further described below. The sample detection area is defined by anoptical transmission port and an optical detection port. In thisembodiment, the optical transmission port is a focusing lens (or anothersuitable optical component) or a window optically after the focusinglens. The optical detection port is a photodetector, or a window beforethe photodetector, facing the lens across the detection region 12. Tipsection 14 further includes a proximal necked-down portion 82 that isinserted into the distal end of an outer tube 16, e.g., a 12-mm diameterstainless steel tube. The outer surface of the necked-down portion 82 isbonded to the inner surface of the distal end of the outer tube 16. Theouter tube 16 is configured to receive an inner tube 32.

Since many bioreactor headplates are equipped with ports for receivingvarious fittings which can be screwed in, fitting 18, optional Teflonwasher 20 and EPDM rubber O-ring 22, or another suitable arrangement,provide a seal on the headplate at the top a bioreactor. In more detail,fitting 18 can be a PG13.5 18 fitting having the standard threadtypically used on bioreactor headplates. Optional Teflon washer 20(which does not provide a seal) can be used as a spacer to ensuresealing on certain bioreactor headplates that may have a deeper threadedsection for the PG13.5 fitting. The EPDM O-ring 22 creates the sealbetween the headplate and the bottom of the Teflon washer 22 as thePG13.5 fitting 18 is tightened.

The dimensions of tip section 14 and the outer tube 16 can be selectedaccording to the size of the reactor. In many situations, thelongitudinal distance between the fitting 18 and the optical detectionregion 12 of the tip section 14 is configured to expose detection area12 to the reactor medium being monitored and specifically a portion ofthat medium that is representative of all of the medium in thebioreactor, rather than possibly unmixed medium along a wall of thereactor. In one illustrative example, the distance between the fitting18 and the optical detection region 12 is at least 1 centimeter (cm),e.g., at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 100 cm. Tip section 14 can be smaller, forminiaturized reactor designs, for instance, or larger, for someindustrial scale applications.

Flange arrangement 24 can be made (entirely or in part) of stainlesssteel and includes upper flange 24 b, for joining electronic housing 26to bottom flange 24 a. The lower end of tip section 14 is capped by plug56, made, for instance, of stainless steel. Rubber (EPDM) O-ring 58provides a leak-tight seal.

Within the distal end of the inner tube 32 is a proximal end 84 of ahousing 36. The housing 36 includes elements such as collimator 38 andan electrical connections, e.g., 3-pin POGO connector 40. For analysis,a light beam (generated by a laser 201 in the controller 200) isdirected through collimator 38 and focused by lens 42, the latter beingseated onto a rubber (EPDM) O-ring 44. From lens 42, typically a moldedaspheric lens, light propagates through the bioreactor medium present insample detection region 12 and reaches the packaged photodiode 46,provided, respectively, with anode, cathode and ground wires 48, 50 and52. These wires serve to transmit signal from the photodiode to a femaleconnector of the tip section 14 to the male connector 40 of the housing36; from there, the signal follows along wires 54, through tube 32,electronics housing 26, cable gland 28 and wire harness 30, for signalanalysis, e.g., by controller 200. In general, during operation, thelight from the tunable laser 201 is coupled into the wire harness 30 andspecifically optical fiber 124 and travels through the inner tube 32 ofbe probe. The light exiting from the fiber is conditioned and collimatedby the collimator 38 and focused by aspheric lens 42 to propagatethrough the sample detection region. The transmitted light is thendetected by the photodiode 46 after being modulated by the sample in thearea 12, which will tend to preferentially absorb some wavelengthsrelative to others.

The length or the wires and/or optical fiber employed can be configuredaccording to the overall size of the probe, which, in turn, can dependon factors such as the size of the reactor being monitored.

The controller 200 monitors the response of the photodiode 46 via theanode wire 48, cathode wire 50, and ground wire 52. The detachableelectrical connections are made through the 3-pin connector 40. Thus,the controller can resolve the absorption spectra of the sample bymonitoring the spectral scanning of the tunable laser over its scan bandrelative to the time-response of the photodiode 46. Generally, thetunable laser or tunable laser system sweeps its narrow band emissionover some region of the electromagnetic spectrum such as the NIR and/orSWIR regions, or portions thereof.

It is possible to convey the transmitted light, e.g., via fiber optics,to a photodetector external to the reactor. However, an arrangement inwhich the detector (photodiode 46 in FIG. 3 ) is part of the in-situprobe exploits signal to noise ratio (SNR) advantages of transmittingelectrical signals, e.g., along wires 48, 50, 52 and 54 (relative todirecting light transmitted through the sample detection region 12 to anexternal photodiode detector, via fiber optics).

Stainless steel set screw 60 secures the collimator 38, while a hollowstainless steel set screw 62 can be employed to apply force to the backof the molded aspheric lens 42 and create a seal with EPDM O-ring 44 andthe stainless steel tip body.

FIGS. 4A and 4B are views of an arrangement suitable for the assembly ofthe housing 36 (including, as already described, collimator 38 and the3-pin POGO connector 40) and the lower tip section 14. The lower tipsection 14 includes sample detection region 12, defined by lens 42 andphotodiode 46. The lower tip section 14 is a housing that can befabricated from molded or additively manufactured plastic or other resinor machined from metal.

For ease of assembly, housing 36 and tip section 14 are provided,respectively, with male alignment features 70 and female alignmentfeatures 72 along with shoulders 74 and grooves 75.

A probe such as described above can involve detachable parts,facilitating autoclave treatment of a portion of the in-situ probe fromwhich optical fiber and electrical wire connections have been removed.For example, shown in FIGS. 5A and 5B is an autoclavable arrangement inwhich electronics housing 26 and the inner tube 32 of the in-situ probe10 (see FIGS. 1 and 2 ) is withdrawn from the outer tube 16 and isreplaced with a stainless steel cap 76 sealing the proximal end of theouter tube 16. In more detail, flange 24 a of flange arrangement 24(FIGS. 1 and 2 ) is covered which stainless steel cap 76, using EPDMrubber O-ring 78 and screws 80.

Once autoclaving is completed, the outer tube 16 and the tip section 14are inserted into the sterile or otherwise controlled environment insidethe bioreactor. Then during operation, the stainless steel cap 76 can beremoved and the in-situ probe 10 can be assembled as illustrated in FIG.6 . Specifically, inner tube 32 with the housing 36 is inserted intoouter tube 16 and flange 24 b is attached to flange 24 a, thus bringingtogether electronics housing 26 and the various components describedwith reference to FIG. 3 . The housing 36 mates with the tip section 14and the male electrical connector 40 of the housing 36 connects to thefemale electrical connector of the tip section 14.

Other embodiments of an in-situ probe can be employed. FIGS. 7 and 8 ,for example, are views of in-situ probe 100, configured for deploying asample detection region 12 within the bioreactor and conducting anin-situ analysis of the reactor contents, using, e.g., NIR spectrometry.

As seen in these figures, in-situ probe 100 includes a tip section 14,comprising an outer tube 16, made, for instance from a 12 mm stainlesssteel tubing, and fiber port housing 120, provided with fiber opticalport adjuster 122, for receiving optical fiber 124. Signal cable andconnector 126, can be attached to or detached from the fiber porthousing, as further described below.

Adjustable PG 13.5 fitting 116 and O-ring 118 can be used to create aseal at an existing port of a bioreactor headplate, as described abovewith reference to elements 18 and 22 in FIGS. 1 and 2 . Since theembodiment illustrated in FIGS. 7 and 8 does not require a spacer, noTeflon washer (such as optional Teflon washer 20 in FIGS. 1 and 2 ) isneeded to fit the probe at the reactor headplate.

Below sample detection region 12, probe 100 terminates with plug 56(made of stainless steel, for example).

FIG. 9 is another embodiment of the probe 100. It includes inner tube32, for optical beam isolation. The inner tube 32 defines an opticalbeam free space path 134 along the centerline of the inner tube 32 forpropagating an electromagnetic energy beam, thus avoiding the need foroptical fiber in the probe. The beam (generated, e.g., by the tunablelaser or another suitable source, not shown in FIG. 9 ) is brought intoinner tube 32 of the in-situ probe via upper section 174 of fiber porthousing 120. Designed to be detachable, upper section 174 fits over alower section 160, connected to the outer tube 16.

For analysis, electromagnetic radiation, e.g., in the NIR frequencyregion, is transmitted along the free space optical beam path 134, inthe direction of the arrow, as illustrated in FIG. 10 . The beam isfocused by aspheric lens 42, supported by lens mount 138. The latter issecured with lens mount screw 140, made, e.g., of stainless steel.

After having passed through sample detection region 12, the transmittedlight reaches photodiode 46. In this example, the photodiode 46 islocated behind a window 142 that defines the distal extent of the sampledetection region 12. The photodiode 46 provides a three-pin photodiodeconnector for photodiode anode wire 48, photodiode cathode wire 50, andphotodiode ground wire 52. The photodiode connector and wires areprotected in leak-tight fashion by fitting plug 56 and rubber (e.g.,EPDM) O-ring 58. Signal exits the in-situ probe via signal cable andconnector 126 and is directed to an analyzer, e.g., to controller 200(FIGS. 1 and 2 ) or component thereof, for processing.

The length of the wires and/or optical fiber employed can be configuredaccording to the overall size of the probe, which, in turn, can dependon factors such as the size of the reactor being monitored.

Relative to an alternative arrangement in which light that has passedthrough the sample detection region 12 is transmitted, via fiber optics,e.g., to an external photodiode detector, an arrangement which includesthe detector (photodiode 46 in FIG. 3 ) as part of the in-situ probe andthus transmits an electrical signal (e.g., to controller 200) can leadto improvements in the SNR.

Probe 100 can involve detachable parts, facilitating autoclave treatmentof a section of the probe from which the connection to fiber optics andto electrical wires has been removed.

Preparing the autoclavable portion of in-situ probe 100 for autoclavingis illustrated with reference to FIGS. 11A and 11B. As seen in thesefigures, the autoclavable portion of in-situ probe 100 does not includecomponents such as section 174 of fiber port housing 120 (see, e.g.,FIGS. 7 and 8 ). Rather, section 160 of optical port housing 120 (FIGS.1-3 ) is covered by stainless steel cap 162, which is supported on arubber (e.g., EPDM) O-ring 164. The cap 162 can be secured usingstainless steel screws 166. Also absent is signal cable and connector126 (FIGS. 1 and 2 ), opening 168 being closed using, for instance EPDMrubber plug 170.

After the autoclave treatment is completed, cap 162 and plug 170 can beremoved.

The assembly of the in-situ probe 100, in preparation for use, isillustrated in FIG. 12 . It involves placing section 174 of optical porthousing 120, over section 160, in the direction of the downward arrow,aligning opening 176 with hole 168, which is threaded to receive end 178of signal cable and connector 126.

The in-situ probe can be employed to monitor and/or control a bioreactorautomatically, using a suitable arrangement or system. In someembodiments this arrangement or system includes a controller, e.g.,controller 200 in FIGS. 1, 2, 7 and 8 ). Part of controller 200 is anarrow band tunable light source 201 such as a tunable laser tointerrogate specific wavelengths or wavelength bands of theelectromagnetic spectrum to perform absorption spectroscopy on thecontents of a reactor. The controller 200 also includes a single boardcomputer, in one example, for monitoring the response of the photodiode46 as a function of the instantaneous wavelength of the tunable laser inorder to resolve the absorption spectrum of the material in the sampledetection region 12.

In operation, the laser source 201 spectrally sweeps through a portionof the wavelength band such as from 780 nanometer (nm) to 2500 nm of theelectromagnetic spectrum, or a portion thereof. This light is suppliedto the probe via the wire harness 30 (FIGS. 1 and 2 ) or optical fiber124 (FIGS. 7 and 8 ) and is directed through the center of the stainlesssteel inner tube 32 (see, for example, FIGS. 2 and 8 ) either in a fiberor a free space beam. Optical elements such as a collimator, lens, etc.are used to form and propagate a beam across the sample detection region12. Light that has passed through the sample detection region isdetected by photodiode 46.

Desired scanning parameters can be set and scanning can be conductedaccording to a suitable scanning program. In one example, the frequencyof measurement is set to about every 5 minutes. In many implementations,the sampling is repeated with any desired frequency over any desiredtime period. For example, sampling is repeated (e.g., at a fewminute-intervals) to monitor the entire reactor process (e.g., for aweek, two weeks, three weeks or longer).

Further aspects of the invention relate to a system in which a reactoris monitored and/or controlled using an in-situ probe such as describedabove.

Any number of reactor types can be employed. For example, shown in FIG.13 is system 300 including a bioreactor 302, monitored by an in-situprobe such as, for instance, probe 10 in FIGS. 1-2 or probe 100 in FIGS.7-8 , and controlled by a controller 200. For illustrative purposes, thereactor 302 in FIG. 13 is a stirred tank reactor, which can be acontinuous, semi-continuous or batch type. Stirred tank reactor 302 isprovided with a motorized impeller 304 and sparger 306. Air is suppliedto the sparger via conduit 308, while gas exits the reactor throughconduit 310. Conduit 312 is used to supply ingredients to the reactor,while product can be collected via conduit 314.

Culture medium 316 is monitored by in-situ probe 10, which is providedwith sample detection region 12 (FIGS. 1 through 3 ) and is secured to aport 322 in the bioreactor headplate 318 using fitting 18 (FIGS. 1 and 2). In operation, a light beam generated by the laser in the controller200 and electrical signal from the photodiode 46 are transmitted to andfrom the probe via wire harness 30. Other process parameters (pH, oxygenlevels, etc.) can be monitored using one or more probes 320 which alsocan be controlled by controller 200 or independently of controller 200.

Shown in FIG. 14 is a diagram of a system 400 in which an in-situ probesuch as probe 10 (FIGS. 1-2 ) or probe 100 (FIGS. 7-8 ), for instance,is inserted to monitor and/or control a rocking bioreactor 402—whichincludes bag 404, supported on a heated plate 406. A typically gentlerocking motion is generated by motorized base 408. The in-situ probe(e.g., probe 100 in FIGS. 7 and 8 ) is inserted via top port 410.Analysis of the bag contents 414, can be performed using controller 200,essentially as described above.

Advantages associated with arrangements that reduce, minimize or preventcell handling (drawing the cells through the pumping system, forexample) are illustrated in FIG. 15 . The data show the impact ofvarious techniques on viable cell densities. A levitating pump, forexample, does not involve much cell touching and yields good cellviability. For cells that are not drawn and circulated in the pumpingsystem, as described herein, results are expected to look very similarto those obtained with the static culture.

The in-situ probe described above can be applied in various situations.In one example, the process parameter monitored is cell growth. Shown inFIG. 16 , for example, are scans of samples of increasing cell densitiesin the NIR wavelengths, leading to increased scatter of the beam andthus an apparent increased absorbance.

FIG. 17 compares the in-situ monitoring of CHO cells grown in abioreactor. From spiking cells and then counting off-line, it ispossible to build a calibration model, which then may be used to monitorthe growth of cells in a more complex bioreactor.

FIG. 18 compares samples of Pichia growing in a shake flask. NIRRINmeasurements were performed with an in-line probe taking samplesautomatically and reading absorbance at approximately 1450 nm and werecompared to off-line measurements from a standard spectrophotometer.

In one example, embodiments of the invention are applied to the field ofcell and gene therapy. Typically, such treatments involve collectingcells from a subject's body, modifying (or reprogramming) the cells andgrowth of these cells to a number suitable for re-implantation.

While cell and gene therapies are expected to expand rapidly in thecoming years, a remaining key challenge for researchers and producers isassessing these complicated, living medicines during manufacturing. Asdeveloped by NIRRIN Bioprocess Analytics, Inc., Billerica, Mass., theuse of NIR laser technology, which has the ability to precisely measurecell growth rates and quantify key metabolites in cell cultures, offersa highly useful mechanism for achieving this goal. Techniques describedabove as well as other NIR approaches can be integrated into complexcell and gene therapy production processes, providing valuable insightinto cellular behavior and phenotypes. An illustration is presentedthrough FIG. 18 , which compares samples of Pichia being grown in ashake flask and studied using NIR measurements performed with an in-lineprobe taking samples automatically and reading absorbance atapproximately 1450 nm with off-line measurements from a standardspectrophotometer.

In a specific example, aspect of the invention can be applied to theproduction of chimeric antigen receptor T (CAR-T) cells. This processbegins with the collection and purification of a patient's ownlymphocytes, which are then genetically engineered to target specificcell surface markers and expanded to create a therapeutic infusionproduct. Analysis of cell growth and density is critical to thisprocess, as the U.S. Food and Drug Administration (FDA) requires eachCAR-T batch to contain a minimum number of cells. In addition,assessment of cellular phenotype via measurement of secretedmetabolites, cytokines, and/or other factors can offer insight into themanufacturing process. Applying techniques described herein, thisinformation can be obtained without the need for manual sampling,increasing efficiency and reducing the risk of contamination. Inaddition to CAR-T therapies, applications can also target the productionof allogeneic CAR-T cells, tumor infiltrating lymphocyte therapies,induced pluripotent stem cell treatments, and other ex vivo cell or genetherapy product.

In sum, procedures and techniques relying on NIR laser technology havethe potential to enter the cell and gene therapy production process andprovide important insight into cell quality and therapy development.From determining cell number and density to precisely measuring secretedfactors of interest, there are a number of valuable uses forbiomanufacturers working on next generation therapies.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A device for monitoring a vessel, the devicecomprising: an outer tube; a tip section at the end of the tube, the tipsection having a sample detection region defined by an opticaltransmission port and an optical detection port, wherein light receivedthrough the optical detection port is detected by a photodetector housedin the tip section and wherein light is transmitted in a free space pathalong a length of the tube to the optical transmission port.
 2. Thedevice as claimed in claim 1, further comprising an inner tube housed inthe outer tube.
 3. The device as claimed in claim 2, wherein the innertube defines the free space path for the light for the opticaltransmission port.
 4. The device as claimed in claim 2, furthercomprising a pin connector system for enabling electrical connections tothe tip section when the inner tube is inserted into the outer tube. 5.The device as claimed in claim 1, wherein the outer tube containselectrical wiring for the photodetector.
 6. The device as claimed inclaim 1, further comprising a housing at a proximal end of the outertube for making electrical connections for the optical detector.
 7. Thedevice as claimed in claim 1, further comprising a fitting on the outertube for sealing with a port of a bioreactor.
 8. The device as claimedin claim 1, wherein the light is generated by a tunable laser thatsweeps a narrow band emission across an infrared spectral band.
 9. Thedevice as claimed in claim 1, further comprising a focusing lens in thetip section for conditioning the light transmitted across the sampledetection region.
 10. A method for monitoring a vessel, the methodcomprising: inserting an outer tube with a tip section into the vessel;transmitting light in a free space path along a length of the outertube; then transmitting the light across a sample detection region ofthe tip section from an optical transmission port to an opticaldetection port; and detecting the light received through the opticaldetection port with a photodetector housed in the tip section.
 11. Themethod of claim 10, further comprising autoclaving the outer tubeincluding the tip section prior to inserting the outer tube into thevessel.
 12. The method of claim 10, further comprising, after insertingthe outer tube into the vessel, inserting an inner tube including ahousing into the outer tube.
 13. A device for monitoring a vessel, thedevice comprising: a removable electronic housing; an outer tubeextending from the electronic housing; a tip section at the end of thetube, the tip section having a sample detection region defined by anoptical transmission port and an optical detection port, wherein lightreceived through the optical detection port is detected by aphotodetector.
 14. The device as claimed in claim 13, wherein thephotodetector is housed in the tip section.
 15. The device as claimed inclaim 13, further comprising a flange arrangement for connecting theelectronic housing to the outer tube.
 16. The device as claimed in claim13, further comprising a cap for sealing a proximal end of the outertube when the removable housing is removed.
 17. The device as claimed inclaim 13, further comprising a flange arrangement on the outer tube foralternatively receiving the cap or the removable electronic housing.